Carrico Zachary M, Le Geneva, Malinow Roberto
Center for Neural Circuits and Behavior, Departments of Neuroscience and Biology, University of California at San Diego, La Jolla, CA 92093, USA.
J Biol Methods. 2017;4(3). doi: 10.14440/jbm.2017.200. Epub 2017 Aug 7.
Robust assays for detecting the effects of elevated concentrations of amyloid-β (Aβ) may facilitate Alzheimer's disease research. An appropriate assay would be high-throughput and enable identification of drugs and genetic mutations that block the effects of Aβ, potentially leading to treatments for Alzheimer's disease. We discovered that the commonly used cytomegalovirus (CMV) enhancer/promoter is sensitive to the effects of Aβ. By combining the CMV enhancer/promoter with a fluorescent protein, we created a reporter system that produces changes in intracellular fluorescence in response to Aβ. Using hippocampal neurons, we quantified the ability of a CMV-fluorescent protein recombinant reporter to detect both exogenously applied and overexpressed Aβ. This is the first report of a high-throughput enhancer/promoter-based Aβ detection method. The reporter is able to detect the effects of elevated concentrations of Aβ in a high-throughput fashion, providing a new tool for Alzheimer's disease research and important knowledge about the commonly used CMV enhancer/promoter.
用于检测淀粉样β蛋白（Aβ）浓度升高所产生影响的稳健检测方法可能会促进阿尔茨海默病的研究。一种合适的检测方法应具有高通量特性，并能够识别出可阻断Aβ作用的药物和基因突变，这有可能带来针对阿尔茨海默病的治疗方法。我们发现常用的巨细胞病毒（CMV）增强子/启动子对Aβ的作用敏感。通过将CMV增强子/启动子与一种荧光蛋白相结合，我们构建了一个报告系统，该系统能响应Aβ产生细胞内荧光变化。利用海马神经元，我们量化了CMV-荧光蛋白重组报告基因检测外源性应用和过表达Aβ的能力。这是基于高通量增强子/启动子的Aβ检测方法的首次报道。该报告基因能够以高通量方式检测Aβ浓度升高所产生的影响，为阿尔茨海默病研究提供了一种新工具，并提供了有关常用CMV增强子/启动子的重要知识。
